Glycosyltransfer mechanism of α-glucosyltransferase from Protaminobacter rubrum

Article abstract of DOI:10.1016/S0008-6215(98)00225-0

α-Glucosyltransferase (αGT) from Protaminobacter rubrum catalyzes either an intramolecular transglucosylation of sucrose, giving isomaltulose, or intermolecular transglucosylations from sucrose to versatile acceptors. To obtain insights into this enzyme,

Full text of DOI:10.1016/S0008-6215(98)00225-0

Carbohydrate Research 312 (1998) 103±115
Glycosyltransfer mechanism of
ꢀ-glucosyltransferase from Protaminobacter
rubrum
Hiroyuki Kakinuma, Hideya Yuasa, Hironobu Hashimoto *
Department of Life Science, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology,
Nagatsuta, Midoriku, Yokohama, 226-8501 Japan
Received 29 May 1998; accepted 3 August 1998
Abstract
ꢀ-Glucosyltransferase (ꢀGT) from Protaminobacter rubrum catalyzes either an intramolecular
transglucosylation of sucrose, giving isomaltulose, or intermolecular transglucosylations from
sucrose to versatile acceptors. To obtain insights into this enzyme, especially in connection with
the glycosyltransfer mechanism, kinetic studies were carried out using synthetic sucrose analogs.
The kcat values obtained for 10-substituted sucrose analogs exhibited a strong correlation with
Hammett substituent constants with a slope of 2, suggesting a rate-limiting glycoside cleavage
with a completely protonated transition state. The ꢀ-deuterium isotope e€ect (kH/kD=1.20‹0.08)
indicated an oxocarbenium ion-like transition state. Nojirimycin and 10-amino-10-deoxy and 30-
amino-30-deoxy analogs of 60-chlorosucrose had relatively strong inhibitory e€ects toward the
intramolecular glucosylation, (inhibition at 10 mM being 97, 71, and 78%, respectively) in contrast
to the 40-amino-40-deoxy and 60-amino-60-deoxy analogs, which showed moderate or no inhibitory
activity. This tendency is presumably due to the presence of carboxylates as catalytic groups of the
enzyme. These results reveal similarities between ꢀGT and some glycosidases in the glycoside
cleavage mechanism. # 1998 Elsevier Science Ltd. All rights reserved
Keywords: Protaminobacter rubrum; ꢀ-Glucosyltransferase; Glucosyltransfer mechanism; 30-Amino-60-chloro-
30,60-dideoxysucrose
1. Introduction
transglucosylation when a 60-blocked sucrose deri-
vative is used as a donor substrate (Scheme 1) [3,4],
allowing ꢀ-d-glucosylation of the hydroxymethyl
group attached to a ®ve-membered ring structure,
such as benzyl arabinofuranoside (2) or 2,3-O-iso-
propylideneglycerol. Depending on substrate and
conditions, ꢀGT hydrolyzes the ꢀ-glucopyranoside
moiety [4,5]. Thus this enzyme can be classi®ed as
an ꢀ-glucosidase that works with retention of the
anomeric con®guration.
ꢀ-Glucosyltransferase (ꢀGT) from Protamino-
bacter rubrum is an industrially important enzyme,
since it produces non-cariogenic isomaltulose
[6-O-(ꢀ-d-glucopyranosyl)-d-fructose] [1,2] from
sucrose by an intramolecular transglucosyla-
tion. This enzyme also catalyzes intermolecular
* Corresponding author. Fax: +81-45-922-2432.
0008-6215/98/$Ðsee front matter # 1998 Elsevier Science Ltd. All rights reserved
PII S0008-6215(98)00225-0

104
H. Kakinuma et al./Carbohydrate Research 312 (1998) 103±115
Scheme 1.
The glycosyl transfer reaction catalyzed by gly-
reported. Indeed, all attempts to obtain the com-
pound 2 by treatment of d-arabinose with acidic
benzyl alcohol ended in a very poor yield. We thus
synthesized compound 2 by inversion of the con-
®guration at C-2 of benzyl ꢁ-d-ribofuranoside (4)
[9]. Protection of the hydroxyl groups at the 3,5-
position of ꢁ-d-ribofuranoside 4 was achieved by
disiloxanylidenation to give compound 5 (Scheme
2). Swern oxidation, followed by reduction with
NaBH4, a€orded inversion of the con®guration at
C-2 to give the arabinofuranoside derivative 6. The
coupling constants for the ring protons of the both
compounds 5 (J1,2=0, J2,3=J3,4=5.3 Hz) and 6
(J1,2=4.6, J2,3=7.3, J3,4=5.6 Hz) suggests a twist
conformation (3T2). Deprotection of the dis-
iloxanylidene group of the compound 6 gave the
desired compound 2.
First attempt at the synthesis of [1-2H]-sucrose
derivative 11 was made by a glucosyl transfer from
uridine 50-(ꢀ-d-[1-2H]-glucopyranosyl diphosphate)
to 6-chloro-6-deoxy-d-fructose using sucrose syn-
thetase. Though this enzyme is known to catalyze
the glucosylation of 1-deoxy-1-¯uoro-d-fructose to
give 10-deoxy-10-¯uorosucrose [10], our substrates
gave no coupling products. Thus we turned our
attention to the reverse reaction catalyzed by
phosphorylase, which has been used for the che-
moenzymatic synthesis of sucrose [11,12]. The sub-
strate phosphate 10 was synthesized by dibenzyl
cosidases has been extensively investigated [6],
partly because understanding of it is important
to improve the glycosyltransfer activity. Indeed,
site-directed mutagenesis based on mechanistic
considerations has allowed a developement of the
glycosidase with high glycosyltransfer acitivity [7].
Since ꢀGT is apparently similar to glycosidases
and its glycosyltransfer activity is potentially
improvable, we feel compelled to investigate its
mechanism. Among a number of experimental
methods to probe the mechanism of enzyme,
kinetic studies with versatile substrates are con-
sidered relatively easy to obtain the information on
the catalytic groups and the structure of transition
states [8]. Thus we examined a Hammett analysis
for the reaction rates (kcat) with respect to 10-sub-
stituents of the sucrose analogs, ꢀ-deuterium iso-
tope e€ect on kcat, and inhibitory e€ects of some
aminodeoxy derivatives of sucrose. Syntheses of
the 10-deuterio-sucrose derivative and the amino-
deoxy sucrose analogs are also reported.
2. Results and discussion
Syntheses of substrates and inhibitors.ÐTo our
knowledge, the synthesis of benzyl ꢁ-d-arabino-
furanoside (2), the glycosyl acceptor, has not been
Scheme 2. (a) 1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane, imidazole, 72%; (b) 1: (COCl)2±Me2SO, Et3N; 2: NaBH4, 82%; (c) 1:
Bu4NF, 2: Py, Ac2O, 93%; (d) NaOMe, 95%.

H. Kakinuma et al./Carbohydrate Research 312 (1998) 103±115
105
phosphorylation [13] of 2,3,4,6-tetra-O-acetyl-d-
[1-2H]-glucopyranose (8) [14], and removal of the
benzyl groups by catalytic hydrogenation (Scheme
3). Transglucosylation of 6-chloro-6-deoxy-d-fruc-
tose with the phosphate 10 gave [1-2H]-60-chloro-
60-deoxysucrose (11), but the yield was poor.
40-Amino-60-chloro-40,60-dideoxysucrose (17) was
synthesized (Scheme 4), according to the synthesis
of 40-acetamido-40-deoxysucrose [15]. The 60-chloro
derivative 13, which was derived from the suitably
protected sucrose derivative 12 [16], was treated
with PPh3 and DEAD, and the O-isopropylidene
groups were substituted with acetyl groups to give
the 30,40-epoxide 15 with a lyxo con®guration at the
fructose moiety. The H NMR spectrum of the
1
compound 15, e.g., J3,4=2.9, J4,5=0 Hz, was very
similar to that reported for the corresponding 60-O-
acetate (J3,4=3.0, J4,5=0.5 Hz) [16], supporting the
assignment. The pentaacetate 15 was treated with
NaN3 to give the 40-azide derivative of 60-chloro-
sucrose 16. The chemical shifts for H-30 and H-40
(ꢂ 5.36, 4.20) and coupling constants (J3,4=J4,5
=7.9 Hz) fully support the assigned structure, as is
the case for the corresponding 60-acetate [15].
Deacetylation of 16, followed by reduction of the
azide group, a€orded the desired compound 17.
Scheme 3. (a) 1: LDA, 2: PO(OBn)2Cl, 53% (ꢀ), 36% (ꢁ); (b) 1: 10% Pd±C/H2, NaHCO3, 2: Dowex 50WÂ8 (Na+ form), 98%; (c)
sucrose phosphorylase, 6-chloro-6-deoxyfructose, 13%.
Scheme 4. (a) PPh3, CCl4, 86%; (b) DEAD, PPh3; (c) 1: 70% AcOH, 2: Py, Ac2O, 53% (from 13); (d) 1: NaN3, NH4Cl, 2: Py,
Ac2O, 84%; (e) 1: NaOMe, 2: Lindlar catalyst/H2, quant.

106
H. Kakinuma et al./Carbohydrate Research 312 (1998) 103±115
Introduction of 30-amino group into the sucrose
the resulting hydroxyl group with azide group were
employed (Scheme 5). Disiloxanylidenation of the
di-O-isopropylidene derivative 12 gave compound
18, in which only the 30-OH is unprotected.
Nucleophilic substitution reaction at C-30 was ®rst
attempted for the 30-O-tri¯uoromethanesulfonate
(30-O-Tf) of compound 18. However, the reaction
derivative was rather problematic, and both the
reductive amination of the 30-ulose derivative and
the nucleophilic ring-opening with NaN3 of the
30,40-epoxide having a ribo con®guration at the
fructose moiety ended in failure. Thus, inversion of
the con®guration at 30-position and substitution of
Scheme 5. (a) 1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane, imidazole, 82%; (b) 1: Py, Tf2O, 2: HF±Py, 3: Py, Ac2O, 86%; (c)
AcOCs, 18-crown-6, 99%; (d) NH3, 0 ꢀC, 71%; (e) 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane, imidazole, 82%; (f) 1: Py, Tf2O, 2:
NaN3, 47%; (g) Bu4NF, 56%; (h) Py, TsCl, 88%; (i) 1: LiCl, 2: 70% AcOH, 3: Py, Ac2O, 82%; (j) 1: NaOMe, 2: Lindlar catalyst/
H2, 52%.

H. Kakinuma et al./Carbohydrate Research 312 (1998) 103±115
107
did not proceed at all, probably because of bulki-
ness of the disiloxanylidene group. Thus the dis-
iloxanylidene group was removed, and acetates
were introduced to the resulting hydroxyl groups
to give compound 19. The series of the 30-O-Tf
derivatives were unexpectedly ꢀstable, and com-
pound 19 could be stored at 4 C over more than
one month. Compound 19 was treated with CsOAc
in the presence of 18-crown-6 [17] a€ording the
compound 20. Selective deprotection of the acetyl
groups at the 30,40,60-positions of 20 with ammonia,
followed by reconstruction of the disiloxanylidene
group at the 40,60-positions, a€orded compound 22.
Nucleophilic substitution reaction at the resulting
30-OH with NaN3, introduction of 60-chloro group
in a usual manner, and deprotection gave the
desired product 27. Stereochemistry of the fructose
moiety of 27 was con®rmed by the large coupling
comparisons of the donor 1 and 28 (Table 1).
When omitting the SH group, a signi®cant corre-
lation was obtained with r and ꢃ values of 0.961
and 2.0, respectively. This strongly negative ꢃ
value suggests a large degree of positive charge
accumulation on the leaving oxygen at the transi-
tion state, and thus a completely protonated tran-
sition state structure (Fig. 3) [19] is indicated. This
suggestion is supported from the negative ꢃ values
reported for the acid-catalyzed hydrolyses of aryl
glycopyranosides [20±22]. Deviation of the SH
substituent from the regression line is presumably
due to an inhibition of protonation of the leaving
oxygen by a hydrogen bonding. Though the Km
values also vary depending on the donor structure,
no clear explanations for this structure±activity
relationship were obtained from available sub-
stituent constants.
0
0
0
0
constants of the ring protons (J3 ,4 =J4 ,5 =9.2 Hz).
Enzymatic study.ÐMichaelis±Menten para-
meters were obtained by Lineweaver±Burk plot for
the intermolecular transglucosylation by ꢀGT with
respect to the donor substrates (1, 28, 29, 30, 31,
32, 33) [5] at a constant concentration of the
acceptor substrate 2 (Table 1). Variation of the
10-substituent signi®cantly a€ects the kcat value
(Table 1), indicating that the formation of the glu-
cosyl±enzyme intermediate (step 1 in Fig. 1) is rate
limiting, because the structures of the liberated
fructose derivatives cannot a€ect the cleavage of
the intermediate (step 2 in Fig. 1). Hammett ana-
lysis was performed for the kcat values with respect
to the inductive e€ect of the 10-substituents (Fig. 2).
The 60-substituents of the donor were determined
to be una€ected to the kinetic constants from
The secondary deuterium kinetic isotope e€ect
measured for the 60-chlorosucrose derivatives 1 and
11 (kH/kD=1.20‹0.08) indicates a glycosyl cation-
like transition state, since the values more than
1.05 re¯ect substantial sp3 to sp2 rehybridization
[23]. By the same token, transition states with
oxocarbenium ion characters have been sug-
gested for an acid-catalyzed hydrolysis of methyl
ꢀ-d-glucopyranoside (kH/kD=1.14) [24], and the
hydrolyses catalyzed by lysozyme (kH/kD=1.11)
[25], exo-ꢀ-glucanase (kH/kD=1.20) [26], sucrase
(kH/kD=1.14) [27], and isomaltase (kH/kD=1.20)
[27].
The characteristics of ꢀGT evaluated above are
similar to those of ꢀ-glucosidases [27±29], whose
activities are inhibited by some azasugars [29,30].
A proposed inhibition mechanism of the azasugars
is that the conjugated acids of the amines bind
tightly to the deprotonated catalytic group. There-
fore, if ꢀGT has an acid catalytic group, nojir-
imycin is considered to inhibit its activity. In
practice, nojirimycin inhibited the activity by 97%
at 10 mM concentration (Table 2). Interestingly,
while 10-amino-10-deoxy (34) [5] and 30-amino-30-
deoxy 27 derivatives of 60-chlorosucrose also
inhibited the activity to a considerable extent, the
40-amino-40-deoxy 17 and 60-amino-60-deoxy (35)
[31] derivatives scarcely inhibited the activity
(Table 2). This is considered due to the presence of
acid catalytic groups, probably carboxylates, near
the reaction center, as proposed to those in most
glycosidases [30]. We thus propose a structure of
the transition state of the transglucosylation cata-
lyzed by ꢀGT as depicted in Fig. 3, where the two
Table 1
Michaelis±Menten parameters for the transglucosylation by
ꢀGT
a
Donor
ꢄI of R1 Km kcat
kcat/Km
1
(mM) (s 1) (s mM
)
1
1 (R1=OH, R2=Cl)
28 (R1=OH, R2=H)
29 (R1=Cl, R2=Cl)
30 (R1=H, R2=H)
31 (R1=SH, R2=Cl)
32 (R1=F, R2=Cl)
33 (R1=OMe, R2=Cl)
0.24
0.24
0.47
0.0
0.27
0.54
0.30
46
43
16
15
0.35
0.35
0.51
0.33
0.30
0.23
0.14
5.9 3.0
22
66
6.1 1.8
8.7 2.0
53
7.3
a
The ꢄI values were taken from Ref. [18].

108
H. Kakinuma et al./Carbohydrate Research 312 (1998) 103±115
Fig. 1. A ping-pong mechanism in the transglucosylation reaction catalyzed by ꢀGT.
carboxylates behave as catalytic groups and one of
them is used for the protonation of the leaving
oxygen.
Benzyl 3,5-O-(1,1,3,3-tetraisopropyldisiloxane-
1,3-diyl)-b-d-ribofuranoside (5).ÐTo a solution of
benzyl ꢁ-d-ribofuranoside (4) [9] (2.54 g,
10.6 mmol) in Me2NCHO (25 mL) were added
imidazole (2.88 g, 42.3 mmol) and 1,3-dichloro-
1,1,3,3-tetraisopropyldisiloxꢀane (3.4 mL, 10.6 mmol)
under Ar atmosphere at 0 C. After 15 h, the mix-
ture was diluted with aq NaHCO3 and extracted
with EtOAc (2Â100 mL). The combined organic
layer was washed with brine, dried (MgSO4), and
evaporated. Flash chromatography (8:1 hexane±
EtOAc) gave 5 (3.69 g, 72%): [ꢀ]2d2 84ꢀ (c 1.5,
3. Experimental
Melting points were measured with a Yanagi-
moto melting point apparatus and are uncorrected.
Optical rotations were determined with a JASCO
DIP-4 polarimeter. NMR spectra were obtained
1
with a JEOL EX-270 spectrometer. In H NMR,
1
chemical shifts were recorded in ppm using Me4Si
(0 ppm in CDCl3) or HDO (4.8 ppm in D2O) as an
internal standard. CDCl3 (77.0 ppm) or dioxane
(64.0 ppm in D2O) was used as an internal standard
in 13C NMR. In 31P NMR, the external standard
H3PO4 was adjusted to 0 ppm. Flash column chro-
matography was carried out on Wako gel C-300
(Wako). Gel ®ltration was performed on Sephadex
G-15 (Pharmacia). Sucrose phosphorylase (EC
2.4.1.7) from Leuconostoc mesenteroides was pur-
chased from Sigma. ꢀ-GT from P. rubrum CBS
574,77 was kindly donated by Mitsui Sugar Co. Ltd.
CHCl3); H NMR (CDCl3): ꢂ 7.40±7.25 (m, 5 H,
Ph), 5.03 (s, 1 H, H-1), 4.68 (d, 1 H, J 11.6 Hz,
PhCH2), 4.57 (t, 1 H, J2,3 5.3, J3,4 5.3 Hz, H-3),
4.44 (d, 1 H, PhCH2), 4.10±4.02 (m, 2 H, H-5a,
5b), 4.09 (d, 1 H, H-2), 3.84±3.76 (m, 1 H, H-4),
2.99 (brs, 1 H), 1.15±0.98 (m, 28 H, CH(CH3)2).
Anal. Calcd for C24H42O6Si2: C, 59.71; H, 8.77.
Found: C, 59.63; H, 8.57.
Benzyl 3,5-O-(1,1,3,3-tetraisopropyldisiloxane-
1,3-diyl)-b-d-arabinofuranoside (6).ÐTo a solu-
tion of (COCl)2 (0.82 mL, 9.6 mmol) in CH2Cl2
(15 mL) was added dropwise a solution of Me2SO
ꢀ
(1.33 mL, 18.7 mmol) in CH2Cl2 (5 mL) at 78 C.
After stirring for 15 min, a solution of 5 (1.52 g,
3.15 mmol) in CH2Cl2 (18 mL) was added. After
stirring at 78 ꢀC for 40 min, Et3N (2.6 mL,
19 mmol) was added dropwise so that the pH of the
solution became 6.5±7. After 5 min, the solution
was warmed up to the room temperature, mixed
Fig. 2. Hammett plot for the transglucosylation reaction of
the 10-substituted sucrose analogs catalyzed by ꢀGT.
Fig. 3. A proposed structure of the transition state.

H. Kakinuma et al./Carbohydrate Research 312 (1998) 103±115
109
Table 2
Inhibitory activity of the aminodeoxysucrose derivatives
toward the conversion of sucrose into isomaltulose by ꢀGT
PhCH2), 4.48 (d, 1 H, PhCH2), 4.43 (dd, 1 H, J4,5a
4.0, J5a,5b 11.6 Hz, H-5a), 4.22 (dd, 1 H, J4,5b
7.3 Hz, H-5b), 4.13 (ddd, 1 H, H-4), 2.10, 2.09, 2.05
(s, each 3 H, OAc). Anal. Calcd for C18H22O8: C,
59.01; H, 6.05. Found: C, 58.72; H, 6.00.
a
Inhibitor
Inhibition (%)
Benzyl b-d-arabinofuranoside (2).ÐTo a solution
of 7 (1.15 g, 3.14 mmol) in dry MeOH (15 mL) was
added NaOMe (10 mg, 0.19 mmol). The solution
was stirred for 24 h and evaporated. Flash chro-
matography (10:1 CHCl3±MeOH) gave a colorless
oil, which crystallized on standing to give 2
Nojirimycin
97
71
78
20
0
34 (R1=NH2, R2=R3=OH, R4=Cl)
27 (R1=R3=OH, R2=NH2, R4=Cl)
17 (R1=R2=OH, R3=NH2, R4=Cl)
35 (R1=R2=R3=OH, R4=NH2)
ꢀ
(715 mg, 95%): mp 72±74 C; [ꢀ]2d2 105ꢀ (c 1.0,
1
H2O); H NMR (D2O): ꢂ 7.50±7.38 (m, 5 H, Ph),
a Inhibition at inhibitor and substrate (sucrose) concentrations
of 10 and 250 mM, respectively.
5.10 (d, 1 H, J1,2 4.3 Hz, H-1), 4.83 (d, 1 H, J
11.6 Hz, PhCH2), 4.62 (d, 1 H, PhCH2), 4.14 (dd, 1
H, J2,3 7.9 Hz, H-2), 4.06 (dd, 1 H, J3,4 6.9 Hz, H-
3), 3.92 (dt, 1 H, J4,5a 3.3, J4,5b 6.9 Hz, H-4), 3.77
(dd, 1 H, J5a,5b 12.2 Hz, H-5a), 3.63 (dd, 1 H, H-
5b); 13C NMR (67 MHz, D2O): ꢂ 137.9, 129.5,
129.3, 129.1, 101.1, 82.9, 77.2, 75.5, 70.5, 64.1.
Anal. Calcd for C12H16O5: C, 59.99; H, 6.71.
Found: C, 59.76; H, 6.75.
with H2O, and extracted twice with CHCl3. The
combined organic layer was dried (MgSO4) and
concentrated to give a colorless oil, which was dis-
solved in EtOH (15 mL). To the solution was
added dropwise a solution of NaBH4 (234 mg,
ꢀ
6.19 mmol) in 2:1 EtOH±H2O (10 mL) at 0 C.
2,3,4,6-Tetra-O-acetyl-a-d-[1-2H]-glucopyrano-
syl dibenzylphosphate (9).ÐTo a solution of 2-pro-
pylamine (0.76 mL, 9.3 mmol) in dry THF (40 mL)
was added 2.5 mL of BuLi (1.6 M in hexane) at
78 ꢀC under Ar atmosphere. After 30 min, a
solution of 2,3,4,6-tetra-O-acetyl-d-[1-2H]-gluco-
pyranose (8) [14] (698 mg, 2.00 mmol) in THF
(30 mL) was added dropwise to the solution over
5 min. The mixture was stirred for 10 min, and
30 mL of dibenzylphosphorochloridate (0.45 M) in
THF solution was added dropwise to the mixture
over 3 min. After 10 min, the resulting mixture was
warmed to room temperature, poured into aq
NaHCO3, and extracted with EtOAc. The organic
layer was washed with brine, dried (MgSO4), and
evaporated. Flash chromatography (1:1 hexane±
EtOAc) gave 9 (649 mg, 53%) and the ꢁ isomer
(439 mg, 36%) as a colorless oil: [ꢀ]2d4 +83ꢀ (c 0.80,
After 15 min, the mixture was neutralized with
30% AcOH, diluted with H2O, and then extracted
with EtOAc. The organic layer was washed with
brine, dried (MgSO4), and evaporated. Flash
chromatography (15:1 hexane±EtOAc) gave 6
(1.25 g, 82%): [ꢀ]2d0 100ꢀ (c 3.2, CHCl3); 1H NMR
(CDCl3): ꢂ 7.40±7.25 (m, 5 H, Ph), 4.97 (d, 1 H, J1,2
4.6 Hz, H-1), 4.77 (d, 1 H, J 11.5 Hz, PhCH2), 4.50
(d, 1 H, PhCH2), 4.26 (dd, 1 H, J2,3 7.3, J3,4 5.6 Hz,
H-3), 4.14 (ddd, 1 H, J2,OH 9.6 Hz, H-2), 3.98 (dd,
1 H, J4,5a 3.0, J5a,5b 9.9 Hz, H-5a), 3.89 (ddd, 1 H,
J4,5b 8.3 Hz, H-4), 3.80 (dd, 1 H, H-5b), 2.31 (d, 1
H, OH), 1.15±0.98 (m, 28 H, CH(CH3)2). Anal.
Calcd for C24H42O6Si2: C, 59.71; H, 8.77. Found:
C, 59.58; H, 8.72.
Benzyl 2,3,4-tri-O-acetyl-b-d-arabinofuranoside
(7).ÐTo a solution of 6 (1.25 g, 2.59 mmol) in
THF (12 mL) ꢀwas added Bu4NF in THF (1 M,
5.2 mL) at 0 C. After 15 min, the mixture was
concentrated and treated with Ac2O (5.0 mL) and
pyridine (10 mL) for 5 h. The solution was poured
into aq NaHCO3 and the mixture was extracted
with EtOAc. The organic layer was washed with
brine, dried (MgSO4), and evaporated. Flash
1
CHCl3); H NMR (CDCl3): ꢂ 7.40±7.31 (m, 10 H,
Ph), 5.49 (dd, 1 H, J2,3 10.2, J3,4 9.6 Hz, H-3), 5.11
(d, 2 H, J 8.6 Hz, PhCH2), 5.10 (dd, 1 H, J4,5 10.2,
H-4), 5.08 (d, 2 H, J 8.6 Hz, PhCH2), 4.98 (dd, 1 H,
JH-P 2.3 Hz, H-2), 4.17 (dd, 1 H, J5,6a 3.9, J6a,6b
12.5 Hz, H-6a), 4.08 (ddd, 1 H, J5,6b 2.3 Hz, H-5),
3.93 (dd, 1 H, H-6b); 31P NMR (D2O): ꢂ 2.1.
Anal. Calcd for C28H32DO13P: C, 55.17; H(D),
5.62. Found: C, 55.24; H(D), 5.68.
chromatography (5:1 hexane±EtOAc) gave
7
(0.88 g, 93%): [ꢀ]2d0 132ꢀ (c 1.4, CHCl3); 1H NMR
(CDCl3): ꢂ 7.38±7.29 (m, 5 H, Ph), 5.40 (dd, 1 H,
J2,3 6.6, J3,4 5.0 Hz, H-3), 5.33 (d, 1 H, J1,2 4.6 Hz,
H-1), 5.06 (dd, 1 H, H-2), 4.78 (d, 1 H, J 11.9 Hz,
a-d-[1-2H]-Glucosylphosphate, disodium salt
(10).ÐA mixture of 9 (672 mg, 1.10 mmol) and
10% Pd±C (30 mg) in 3:2 MeOH±10% NaHCO3

110
H. Kakinuma et al./Carbohydrate Research 312 (1998) 103±115
(30 mL) was stirred under 1 atm of hydrogen gas.
After 2.5 h, the suspension was ®ltered through a
Celite pad and evaporated. The residue was dis-
solved in 13:6:1 MeOH±H2O±Et3N (10 mL), and
the solution was stirred for 4.5 h and evaporated.
The residue was passed through a column of
Dowex 50WÂ8 (Na+ form, 2Â13 cm). After lyo-
philization, the residue was puri®ed by gel ®ltration
(1.7Â100 cm) to give 10 (330 mg, 98%) as a white
chromatography (3:1 EtOAc±hexane) gave 13
(1.19 g, 86%). The analytical sample was prepared
by recrystallization from CH2Cl2±hexane: mp 127±
132 ꢀC; [ꢀ]1d5 +32ꢀ (c 1.0, CHCl3); 1H NMR
(CDCl3): ꢂ 6.11 (d, 1 H, J1,2 4.0 Hz, H-1), 5.25 (t, 1
H, J2,3 9.6, J3,4 9.6 Hz, H-3), 4.23±4.18 (m, 1 H, H-
40), 4.14 (d, 1 H, J1 a,1 b 12.2 Hz, H-10a), 4.10 (q, 1
0
0
H, J4 ,5 6.6, J5 ,6 6.6 Hz, H-50), 3.90±3.70 (m, 8 H,
H-2, 4, 5, 30, 6, 60), 3.04 (d, 1 H, H-10b), 3.08±3.02
(m, 2 H, OH), 2.07 (s, 3 H, OAc), 1.47, 1.46, 1.40,
1.31 (s, each 3 H, CMe2); Anal. Calcd for
C20H31ClO11: C, 49.74; H, 6.47. Found: C, 50.34;
H 6.63.
0
0
0
0
1
solid. H, 13C, and 31P NMR indicated high purity
1
of this compound: H NMR (D2O): ꢂ 3.91 (ddd, 1
H, J4,5 10.2, J5,6a 2.3, J5,6b 5.3 Hz, H-5), 3.88 (dd, 1
H, J6a,6b 12.2 Hz, H-6a), 3.78 (dd, 1 H, J2,3 9.9, J3,4
9.2 Hz, H-3), 3.76 (dd, 1 H, H-6b), 3.51 (dd, 1 H,
JH-P 2.0 Hz, H-2), 3.42 (dd, 1 H, H-4); 13C NMR
(67 MHz, D2O): ꢂ 73.5, 73.1, 72.3 (d, JC-P 7.3 Hz),
70.2, 61.2; 31P NMR (D2O): ꢂ 1.6.
10,2,3,4,6-Penta-O-acetyl-30,40-anhydro-60-chloro-
60-deoxy-tagato-sucrose (15).ÐTo a solution of 13
(1.21 g, 2.50 mmol) in toluene (11 mL) were added
DEAD (0.80 mL, 5.09 mmol) and triphenylpho-
sphine (1.70 g, 6.49 mmol) under Ar atmosphere at
[1-2H]-60-Chloro-60-deoxysucrose (11).ÐTo
a
ꢀ
solution of 6-chloro-6-deoxyfructose (215 mg,
1.08 mmol) in 25 mM citrate bu€er (pH 6.8,
4.13 mL) were added 10 (330 mg, 1.08 mmol) and
sucrose phosphorylase (9.7 mg, 49 U), and the
0 C. The mixture was kept for 1.5 h at room tem-
perature. After MeOH (5 mL) was added, the mix-
ture was stirred for 20 min and diluted with EtOAc.
The solution was washed with brine, dried
(MgSO4), and evaporated. Flash chromatography
(2:1 EtOAc±hexane) gave a syrupy 14 (1.10 g). {1H
NMR (CDCl3): ꢂ 5.87 (d, 1 H, J1,2 3.6 Hz, H-1),
5.30 (dd, 1 H, J2,3 9.6, J3,4 9.6 Hz, H-3), 4.32 (q, 1
ꢀ
mixture was incubated at 30 C. After 1 h, the
enzyme (8.4 mg, 42 U) was further added into the
mixture, and the reaction mixture was incubated
for an additional 1 h. After boiling for 15 min, the
mixture was ®ltered through a membrane and lyo-
philized. The residue was puri®ed by ¯ash chro-
matography (gradient from 20:1 EtOAc±EtOH to
6:2:1 EtOAc±EtOH±H2O) to give 11 (49 mg, 13%):
[ꢀ]2d7 +60ꢀ (c 1.0, H2O); Rf 0.38 (6:2:1 EtOAc±
H, J4 ,5 7.3, J5 ,6 7.3 Hz, H-50), 4.04 (d, 1 H, J1 a,1 b
11.9 Hz, H-10a), 3.95±3.87 (m, 3 H), 3.79 (dd, 1 H,
H-2), 3.66 (t, 1 H, J4,5 9.6 Hz, H-4), 3.65±3.57 (m, 4
H, H-30, 40, 60), 3.42 (d, 1 H, H-10b), 2.07 (s, 3 H,
OAc), 1.46, 1.45, 1.38, 1.35 (s, each 3 H, CM2).}
Compound 14 (1.10 g) was dissolved in 70% AcOH
0
0
0
0
0
0
EtOH±H2O); 1H NMR (D2O): ꢂ 4.24 (d, 1 H, J3 ,4
0
0
8.3 Hz, H-30), 4.13 (t, 1 H, J4 ,5 8.3 Hz, H-40), 4.04
(10 mL), and the mixture was heated at 70 C for
ꢀ
0
0
(dt, 1 H, J5 ,6 5.6 Hz, H-50), 3.92±3.82 (m, 4 H, H-
6,60), 3.76 (t, 1 H, J2,3 9.6, J3,4 9.6 Hz, H-3), 3.71 (s,
2 H, H-10), 3.56 (d, 1 H, H-2), 3.43 (t, 1 H, J4,5
9.6 Hz, H-4); 13C NMR (67 MHz, D2O): ꢂ 104.6,
81.5, 77.1, 76.7, 73.3, 71.7, 70.2, 61.8, 61.1, 45.9.
HRFABMS: Anal. Calcd for C12H20ClDNaO10
(M+Na+) 384.0784, Found 384.0808. Anal. Calcd
for C12H20DO10: C, 39.84; H(D), 6.13. Found: C,
39.49; H(D), 5.95.
20 min. After cooling to room temperature, the
mixture was evaporated. To a solution of the resi-
due in pyridine (7 mL) was added Ac2O (3 mL).
After 3 h, the mixture was poured into aq NaHCO3
and extracted with EtOAc. The organic layer was
washed with aq NaHCO3 and brine, dried
(MgSO4), and evaporated. Flash chromatography
(1:1 hexane±EtOAc) gave 15 (738 mg, 53%): mp
0
0
126±127 C; [ꢀ]1d5 +71ꢀ (c 1.1, CHCl3); H NMR
(CDCl3): ꢂ 5.78 (d, 1 H, J1,2 3,6 Hz, H-1), 5.46 (t, 1
H, J2,3 9.9, J3,4 9.9 Hz, H-3), 5.07 (t, 1 H, J4,5
9.9 Hz, H-4), 4.77 (dd, 1 H, H-2), 4.30 (d, 1 H,
ꢀ
1
3-O-Acetyl-60-chloro-60-deoxy-10,2:4,6-di-O-iso-
propylidenesucrose (13). To a solution of 12 [16]
(1.33 g, 2.87 mmol) in pyridine (15.7 mL) were
added CCl4 (3.1 mL, 32 mmol) and triphenylpho-
J1 a,1 b 11.9 Hz, H-10a), 4.32±4.07 (m, 4 H), 4.13 (d,
0
0
ꢀ
0 0
sphine (1.56 g, 5.95 mmol). After 30 min at 60 C,
1 H, H-10b), 3.89, 3.73 (each d, each 1 H, J3 ,4
MeOH (10 mL) was added to the mixture and the
mixture was stirred for 5 min at the same tempera-
ture. After cooling to room temperature, the mixture
was diluted with EtOAc. The solution was washed
with brine, dried (MgSO4), and evaporated. Flash
2.9 Hz, H-30 or 40), 3.59 (dd, 1 H, J5 ,6 a 8.6, J6 a,6 b
0 0 0 0
10.9 Hz, H-60a), 3.52 (dd, 1 H, J5 ,6 b 5.9 Hz, H-60b),
2.13, 2.08, 2.04, 2.02 (each s, 15 H, OAc). Anal.
Calcd for C22H29ClO14: C, 47.79, H 5.29; Found:
C, 47.55; H 5.32.
0
0

H. Kakinuma et al./Carbohydrate Research 312 (1998) 103±115
111
ꢀ
10,2,3,30,4,6,-Hexa-O-acetyl-40-azido-60-chloro-
40,60-dideoxysucrose (16).ÐTo a solution of 15
(384 mg, 0.695 mmol) in 8:1 EtOH±H2O (12 mL)
were added NaN3 (348 mg, 5.35 mmol) and NH4Cl
(348 mg, 6.50 mmol), and the mixture was stirred
at 0 C. After 1 h, aq NaHCO3 was added to the
mixture, and it was extracted with EtOAc
(2Â50 mL). The combined organic layer was
washed with brine, dried (MgSO4), and evapo-
rated. Flash chromatography (6:1 hexane±EtOAc)
gave 18 (4.35 g, 82%): [ꢀ]2d3 +3.3ꢀ (c 1.1, CHCl3);
1H NMR (CDCl3): ꢂ 5.99 (d, 1 H, J1,2 4.0 Hz, H-1),
5.20 (t, 1 H, J2,3 9.6, J3,4 9.6 Hz, H-3), 4.25 (dd, 1
ꢀ
for 73 h at 80 C. After cooling to room tempera-
ture, the mixture was evaporated to give a residue
that was acetylated with a mixture of pyridine
(7 mL) and Ac2O (3 mL). The reaction mixture was
poured into aq NaHCO3 and extracted with
EtOAc. The organic layer was washed with aq
NaHCO3 and brine, dried (MgSO4), and evapo-
rated. Flash chromatography (1:1 hexane±EtOAc)
H, J3 ,4 7.3, J4 ,5 5.3 Hz, H-40), 4.10 (d, 1 H, J1 a,1 b
12.5 Hz, H-10a), 3.98 (d, 1 H, H-30), 3.93±3.60 (m, 8
H, H-2, 4, 5, 50, 6, 60), 3.44 (d, 1 H, H-10b), 2.06 (s,
1 H, OAc), 1.47, 1.44, 1.41, 1.30 (each s, each 3H,
CMe2), 1.13±1.02 (m, 28 H, CH(CH3)2). 13C NMR
(67 MHz, CHCl3): ꢂ 102.7, 101.2, 99.6, 91.1, 81.7,
80.8, 80.3, 71.5, 70.4, 66.1, 65.7, 64.1, 62.2, 28.9,
25.3, 23.9, 20.9, 18.9, 17.5, 17.4, 17.3, 17.0, 13.4,
13.2, 13.0, 12.7, 12.5. Anal. Calcd for C32H58
O13Si2: C, 54.37; H, 8.27. Found: C, 54.47; H, 8.28.
3,40,60-Tri-O-acetyl-10,2:4,6-di-O-isopropylidene-
30-O-tri¯uoromethanesulfonylsucrose (19).ÐTo a
solution of pyridine (2.0 mL, 25 mmol) and tri-
¯uoromethanesulfonic anhydride (2.0 mL, 12 mmol)
in CH2Cl2 (43 mL) was added dropwise a solution
ofꢀ 18 (4.35 g, 6.15 mmol) in CH2Cl2 (40 mL) at
0 C. After 1 h, the mixture was poured into aq
NaHCO3 and extracted with CH2Cl2. The organic
layer was dried (MgSO4) and evaporated. The
residue was dissolved in pyridine (20 mL) and the
mixture was transferred into a Te¯on tube. To
the mixture was added HF±pyridine solution
(5 mL) at 0 ꢀC. After 15 min, the mixture was
poured into aq NaHCO3 and extracted with
EtOAc. The organic layer was washed with brine,
dried (MgSO4), and evaporated. The residue was
dissolved in pyridine (10 mL) and Ac2O (5 mL).
After 3 h, the mixture was poured into aq NaHCO3
and extracted with EtOAc. The organic layer was
washed with brine, dried (MgSO4), and evapo-
rated. The residue was crystallized from hexaneꢀto
give 19 as a white solid (3.62 g, 86%): mp 133 C
(dec.); [ꢀ]2d1 +13ꢀ (c 1.2, CHCl3); 1H NMR
(CDCl3): ꢂ 6.09 (d, 1 H, J1,2 3,6 Hz, H-1), 5.45 (dd,
0
0
0
0
0
0
ꢀ
gave 16 (373 mg, 84%): mp 83±85 C; [ꢀ]1d7 +45ꢀ
1
(c 1.1, CHCl3); H NMR (CDCl3): ꢂ 5.57 (d, 1 H,
J1,2 3.6, H-1), 5.43 (t, 1 H, J2,3 9.9, J3,4 9.9 Hz, H-
3), 5.36 (d, 1 H, J3 ,4 7.9 Hz, H-30), 5.07 (t, 1 H, J4,5
9.9 Hz, H-4), 4.90 (dd, 1 H, H-2), 4.30±4.11 (m,
0
0
4H, H-5, 50, 6), 4.20 (t, 1 H, J4 ,5 7.9 Hz, H-4 ), 4.19
0
0
0
(d, 1 H, J1 a,1 b 11.9 Hz, H-10a), 4.04 (d, 1 H, H-
0
0
10b), 3.79 (d, 1 H, J5 ,6 a 5.3, J6 a,6 b 10.9 Hz, H-60a),
0
0
0
0
3.73 (dd, 1 H, J5 ,6 b 5.3 Hz, H-60b), 2.24, 2.12, 2.11,
2.10, 2.06, 2.02 (each s, 18 H, OAc). 13C NMR
(67 MHz, CHCl3): ꢂ 170.6, 169.9, 169.7, 169.5,
102.9, 90.0, 79.8, 70.1, 69.5, 68.6, 68.5, 65.0, 63.6,
62.5, 43.3, 20.7, 20.6, 20.5. Anal. Calcd for
C24H32ClN3O15: C, 45.18; H, 5.06; N 6.59. Found:
C, 45.09; H, 4.97; N 6.53.
0
0
40-Amino-60-chloro-40,60-dideoxysucrose (17).Ð
To a solution of 16 (285 mg, 0.447 mmol) in dry
MeOH (6 mL) was added NaOMe (7.0 mg,
0.13 mmol), and the mixture was stirred for 2 h. To
the mixture was added Lindlar catalyst (100 mg),
and the mixture was stirred under 1 atm of hydro-
gen for 2 h. The mixture was ®ltered and evapo-
rated. The residue was puri®ed by gel ®ltration and
lyophilized to give 17 (161 mg, 100%) as a white
solid: [ꢀ]1d7 +71 (c 0.90, H2O); H NMR (D2O): ꢂ
ꢀ
1
0
0
5.43 (d, 1 H, J1,2 3.9 Hz, H-1), 4.52 (d, 1 H, J3 ,4
9.6 Hz, H-30) 4.39±4.10 (m, 1 H, H-50), 3.97±3.72
(m, 10 H), 3.59 (dd, 1 H, J2,3 9.9 Hz, H-2), 3.43 (t, 1
H, J3,4 9.9, J4,5 9.9 Hz, H-4). 13C NMR (67 MHz,
D2O): ꢂ 105.1, 93.5, 78.6, 74.8, 73.6, 73.2, 71.7,
70.3, 61.3, 61.2, 57.6, 45.2. Anal. Calcd for C12H22
ClNO9: C, 40.06; H, 6.16; N, 3.89. Found: C,
39.92; H, 6.14; N, 3.85.
1 H, J3 ,4 5.9, J4 ,5 4.3 Hz, H-40), 5.23 (t, 1 H, J2,3
9.6, J3,4 9.6 Hz, H-3), 4.94 (d, 1 H, H-30), 4.32±4.24
0
0
0
0
(m, 2 H, H-60), 4.16 (d, 1 H, J1 a,1 b 12.2 Hz, H-10a),
3.96 (dd, 1 H, J5,6a 5.0, J6a,6b 10.2 Hz, H-6a), 3.90±
3.80 (m, 1 H, H-5), 3.86 (dd, 1 H, H-2), 3.69 (dd, 1
H, J5,6b 3.3 Hz, H-6b), 3.67 (t, 1 H, J4,5 9.9 Hz, H-
4), 3.49 (d, 1 H, H-10b), 2.14, 2.05, 2.04 (each s,
each 3 H, OAc), 1.47, 1.46, 1.41, 1.30 (each s, each
3 H, CMe2). 13C NMR (67 MHz, CHCl3): ꢂ 170.4,
169.6, 169.5, 102.9, 101.7, 99.7, 91.8, 85.9, 79.1,
0
0
3-O-Acetyl-10,2:4,6-di-O-isopropylidene-40,60-O-
(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)sucrose
(18).ÐTo a solution of 12 (3.50 g, 7.54 mmol) in
Me2NCHO (35 mL) were added imidazole (2.2 g,
32 mmol) and 1,3-dichloro-1,1,3,3-tetraisopropyl-
disiloxane (2.4 mL, 7.5 mmol) under Ar atmosphere

112
H. Kakinuma et al./Carbohydrate Research 312 (1998) 103±115
76.5, 71.7, 71.2, 70.3, 64.9, 64.6, 64.4, 61.9, 28.9,
25.3, 23.7, 20.9, 20.7, 20.4, 18.9, 16.8, 12.5. Anal.
Calcd for C25H35F3O16S: C, 44.12; H, 5.18. Found:
C, 44.10; H, 5.38.
99.8, 91.3, 85.5, 77.2, 71.3, 71.2, 70.7, 68.8, 65.0,
64.0, 61.8, 61.2, 28.9, 25.3, 24.2, 21.0, 18.9. Anal.
Calcd for C20H32O12: C, 51.72; H, 6.94. Found: C,
51.67; H, 7.01.
3,30,40,60-Tetra-O-Acetyl-10,2:4,6-di-O-isopropyl-
idene-psico-sucrose (20).ÐTo a solution of 19
(3.60 g, 5.29 mmol) in dry Me2NCHO (36 mL) were
added AcOCs (3.00 g, 15.6 mmol) and 18-crown-6
(4.20 g, 15.9 mmol), and the solution was heated at
3-O-Acetyl-10,2:4,6-di-O-isopropylidene-40,60-O-
(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-psico-
sucrose (22).ÐThe compound 21 (2.02 g,
4.35 mmol) was silylated in the similar manner as
described for the synthesis of 18 to give 22 (2.52 g,
82%) as amorphous: [ꢀ]2d3 +11ꢀ (c 1.2, CHCl3); 1H
NMR (CDCl3): ꢂ 5.91 (d, 1 H, J1,2 4.0 Hz, H-1),
5.22 (t, 1 H, J2,3 9.6, J3,4 9.6 Hz, H-3), 4.59 (dd, 1
ꢀ
95 C for 3 h. After cooling to room temperature,
the mixture was diluted with EtOAc (150 mL) and
extracted with water. The organic layer was
washed with brine, dried (MgSO4), and evapo-
rated. Flash chromatography (2:1 hexane±EtOAc)
gave 20 (3.10 g, 99%) as a white solid: mp 172±
174 ꢀC; [ꢀ]2d3 +28ꢀ (c 1.1, CHCl3); 1H NMR
(CDCl3): ꢂ 6.04 (d, 1 H, J1,2 3.6 Hz, H-1), 5.40 (d, 1
H, J3 ,4 5.0, J4 ,5 5.9 Hz, H-40), 4.15±4.03 (m, 2 H),
0
0
0
0
4.07, (d, 1 H, J1 a,1 b 12.4 Hz, H-10a), 4.05 (d, 1 H,
H-30), 3.81 (d, 1 H, H-10b), 3.81±3.64 (m, 5 H), 3.79
(dd, 1 H, H-2), 3.60 (t, 1 H, J4,5 9.6 Hz, H-4), 2.06
(s, 3 H, OAc), 1.46, 1.44, 1.40, 1.29 (each s, each 3
H, CMe2), 1.16±0.91 (m, 28 H, CH(CH3)2). 13C
NMR (67 MHz, CHCl3): ꢂ 170.1, 107.2, 101.0,
99.6, 91.2, 83.8, 75.8, 71.6, 70.7, 66.2, 64.3, 63.9,
62.3, 29.0, 25.4, 24.0, 21.0, 18.9, 17.5, 17.3, 17.2,
17.0, 13.2, 13.0, 12.8, 12.6. Anal. Calcd for
C32H58O13Si2: C, 54.37; H, 8.27. Found: C, 54.31;
H, 8.67.
0
0
H, J3 ,4 4.9 Hz, H-30), 5.40 (dd, 1 H, J4 ,5 8.5 Hz,
H-40), 5.22 (t, 1 H, J2,3 9.3, J3,4 9.3 Hz, H-3), 4.46±
0
0
0
0
4.39 (m, 1 H, H-50), 4.32 (dd, 1 H, J5 ,6 a 4.6, J6 a,6 b
11.5 Hz, H-60a), 4.20 (dd, 1 H, J5 ,6 b 7.9 Hz, H-60b),
0
0
0
0
0
0
3.95 (d, 1 H, J1 a,1 b 12.2 Hz, H-10a), 3.89 (dd, 1 H,
J5,6a 5.0, J6a,6b 9.6 Hz, H-6a), 3.81 (dd, 1 H, H-2),
3.78 (dd, 1 H, J5,6b 4.6 Hz, H-6b), 3.65 (d, 1 H, H-
10b), 3.62 (dd, 1 H, J4,5 9.9 Hz, H-4), 2.13, 2.06,
2.05, 2.04 (each s, each 3 H, OAc), 1.46, 1.44, 1.38,
1.28 (each s, each 3 H, CMe2). 13C NMR (67 MHz,
CHCl3): ꢂ 170.5, 170.0, 169.2, 169.0, 106.9, 101.3,
99.6, 91.4, 79.6, 75.5, 72.3, 71.6, 71.4, 70.6, 65.0,
64.0, 63.9, 62.1, 29.0, 25.4, 24.0, 20.9, 20.8, 20.4,
18.9. Anal. Calcd for C26H38O15: C, 52.88; H, 6.49.
Found: C, 52.68; H, 6.56.
0
0
3-O-Acetyl-30-azido-30-deoxy-10,2:4,6-di-O-iso-
propidene-40,60-O-(1,1,3,3-tetraisopropyldisiloxane-
1,3-diyl)sucrose (23).ÐInto a solution of pyridine
(1.1 mL, 14 mmol) and tri¯uoromethanesulfonic
anhydride (1.1 mL, 6.5 mmol) in CH2Cl2 (25 mL)
was added dropwise a solution of 22 (2.53 g,
3.58 mmol) in CH2Cl2 (25 mL) at 0 ꢀC. After
15 min, the mixture was poured into aq NaHCO3
and extracted with CH2Cl2. The organic layer was
washed with brine, dried (MgSO4), and evaporated
to dryness. The residue was dissolved in
Me2NCHO (25 mL), NaN3 (726 mg, 11.2 mmol)
and 18-crown-6 (2.8 g, 11 mmol) were addedꢀto the
solution, and the mixture was stirred at 90 C for
40 min. After cooling to room temperature, the
mixture was diluted with EtOAc, washed with
water and brine, dried (MgSO4), and evaporated.
Flash chromatography (6:1 hexane±EtOAc) gave
23 (1.23 g, 47%) as a white solid: [ꢀ]2d2 +29ꢀ (c 2.3,
3-O-Acetyl-10,2:4,6-di-O-isopropylidene-psico-
sucrose (21).ÐInto a solution of 20 (3.10 g,
5.25 mmol) in dry MeOH (97 mL) was bubbled
ꢀ
NH3 (g) for 15 min at 20 C. After completion
of the reaction (4 h) was con®rmed by TLC,
the mixture was evaporated and the residue was
co-evaporated with MeOH. Flash chromatography
(50:1 EtOAc±EtOH) gave 21 (1.73 g, 71%) as a col-
orless oil: [ꢀ]d23 +31ꢀ (c 1.1, CHCl3); Rf 0.63 (10:1
1
EtOAc±EtOH); H NMR (CDCl3): ꢂ 6.19 (d, 1 H,
J1,2 4.0 Hz, H-1), 5.22 (t, 1 H, J2,3 9.2, J3,4 9.2 Hz,
H-3), 4.83±4.76 (m, 1 H, H-40), 4.19±4.11 (m, 2 H,
CHCl3); H NMR (CDCl3): ꢂ 5.94 (d, 1 H, J1,2
4.0 Hz, H-1), 5.28 (t, 1 H, J2,3 9.6, J3,4 9.6 Hz, H-3),
1
H-30, 50), 4.09 (d, 1 H, J1 a,1 b 12.8 Hz, H-10a), 3.97
0
0
(brd, 1 H, J6 a,6 b 12.8 Hz, H-60a), 3.87 (d, 1 H, H-
10b), 3.83 (dd, 1 H, H-2), 3.87±3.77 (m, 2 H, H-5,
60b), 3.64 (dd, 1 H, J4,5 9.9 Hz, H-4), 3.68±3.58 (m,
2 H, H-6), 3.38, 3.13 (each brs, each 1 H, OH), 3.02
(brd, 1 H, J 10.6 Hz, OH), 2.08 (s, 3 H, OAc), 1.47,
1.45, 1.38, 1.34 (each s, each 3 H, CMe2). 13C
NMR (67 MHz, CHCl3): ꢂ 170.5, 107.4, 101.5,
4.60 (dd, 1 H, J3 ,4 8.3, J4 ,5 5.3 Hz, H-40), 4.11 (d,
0
0
0
0
0
0
1 H, J1 a,1 b 12.2 Hz, H-10a), 4.03±3.91 (m, 3 H, H-
50, 60), 3.82 (dd, 1 H, H-2), 3.72 (dd, 1 H, J5,6b 4.3,
J6a,6b 10.2 Hz, H-6a), 3.85±3.78 (m, 2 H, H-5, 6b),
3.63 (t, 1 H, J4,5 9.6 Hz, H-4), 3.40 (d, 1 H, H-10b),
3.29 (d, 1 H, H-30), 2.06 (s, 3 H, OAc), 1.46, 1.45,
1.39, 1.31 (each s, each 3 H, CMe2), 1.14±0.93 (m,
0
0

H. Kakinuma et al./Carbohydrate Research 312 (1998) 103±115
113
28 H, CH(CH3)2). 13C NMR (67 MHz, CHCl3): ꢂ
169.8, 104.5, 101.2, 99.5, 91.4, 82.7, 71.7, 71.4,
70.6, 69.2, 66.0, 65.7, 64.4, 62.2, 28.9, 25.4, 23.9,
20.9, 19.0, 17.6, 17.5, 17.3, 17.0, 16.9, 16.8, 13.4,
13.2, 13.0, 12.8, 12.5. Anal. Calcd for C32H57
N3O12Si2: C, 52.51; H ,7.85. Found: C, 52.82; H,
8.35.
OAc), 1.46, 1.43, 1.38, 1.30 (each s, each 3 H,
CMe2). 13C NMR (67 MHz, CHCl3): ꢂ 170.0,
145.0, 132.5, 129.8, 127.9, 105.5, 101.4, 99.6, 98.7,
91.3, 79.7, 74.4, 71.5, 71.3, 70.7, 67.7, 65.4, 64.3,
62.0, 28.9, 25.2, 23.8, 21.6, 20.9, 18.9, Anal. Calcd
for C27H37N3O13S: C, 50.38; H, 5.79; N, 6.53.
Found: C, 50.34; H, 5.45; N, 6.30.
3-O-Acetyl-30-azido-30-deoxy-10,2:4,6-di-O-iso-
propylidenesucrose (24).ÐTo a solution of 23
(1.17 g, 1.60 mmol) in THF (11 mL) was added ꢀa 1
M solution of Bu4NF in THF (3.2 mL) at 0 C.
After 10 min, the mixture was diluted with EtOAc,
and the solution was washed with water, brine,
dried (MgSO4), and evaporated. Flash chromato-
graphy (2:1 EtOAc±hexane) gave 24 (440 mg, 56%)
as a white solid: mp 193±196 ꢀC; [ꢀ]d22 +17ꢀ (c 1.0,
10,2,3,4,40,6-Hexa-O-acetyl-30 -azido-60-chloro-
30,60-dideoxysucrose (26).ÐTo a solution of 25
(382 mg, 0.593 mmol) in Me2NCHO (4ꢀ.0 mL) was
added LiCl (142 mg, 3.35 mmol) at 80 C for 12 h
under an Ar atmosphere. After cooling to room tem-
perature, the mixture was diluted with EtOAc and
washed with water and brine. The organic layer
was dried (MgSO4) and evaporated. The residue
was dissolved in 70% AcOH (5 mL), and the solu-
tion was stirred at 80 ꢀC. After 2 h, the mixture was
evaporated, and the residue was treated with pyr-
idine (2 mL) and Ac2O (1 mL). The mixture was
poured into aq NaHCO3 and extracted with
EtOAc. The organic layer was washed with brine,
dried (MgSO4), and evaporated. Flash chromato-
graphy (2:1 hexane±EtOAc) gave 26 (309 mg, 82%)
1
CHCl3); H NMR (CDCl3): ꢂ 6.20 (d, 1 H, J1,2
4.0 Hz, H-1), 5.30 (dd, 1 H, J2,3 9.2, J3,4 9.6 Hz, H-
3), 4.93 (dd, 1 H, J3 ,4 9.2, J4 ,5 7.6 Hz, H-40), 4.14
0
0
0
0
(d, 1 H, J1 a,1 b 12.2 Hz, H-10a), 4.03 (dt, 1 H, J5 ,6
0
0
0
0
1.5 Hz, H-50), 3.97±3.80 (m, 3 H, H-5, 60), 3.88 (d, 1
H, H-2), 3.66 (dd, 1 H, J4,5 9.2 Hz, H-4), 3.65±3.58
(m, 2 H, H-6), 3.46 (d, 1 H, H-10b), 3.26 (d, 1 H, H-
30), 3.13±3.00 (m, 1 H, OH), 2.92 (brs, 1 H, OH),
2.08 (s, 3 H, OAc), 1.47, 1.45, 1.38, 1.34 (each s,
each 3 H, CMe2). 13C NMR (67 MHz, CHCl3): ꢂ
169.8, 105.1, 101.8, 99.7, 91.5, 83.9, 71.3, 71.1,
70.5, 69.6, 67.6, 66.1, 64.8, 61.8, 60.8, 28.9, 25.2,
24.0, 20.9, 18.9. Anal. Calcd for C20H31N3O11: C,
49.08; H, 6.38; N, 8.58. Found: C, 48.90; H, 6.46;
N, 8.31.
ꢀ
as crystals: mp 137±139 C; [ꢀ]2d2 +39ꢀ (c 0.60,
1
CHCl3); H NMR (CDCl3): ꢂ 5.57 (d, 1 H, J1,2
4.0 Hz, H-1), 5.48 (dd, 1 H, J2,3 10.3, J3,4 9.9 Hz,
H-3), 5.40 (dd, 1 H, J3 ,4 8.6, J4 ,5 7.3 Hz, H-40),
5.04 (t, 1 H, J4,5 9.9 Hz, H-4), 5.00 (dd, 1 H, H-2),
4.27±4.17 (m, 2 H, H-5, 50), 4.18±4.16 (m, 2 H, H-
0
0
0
0
60), 4.12 (d, 1 H, J1 a,1 b 7.6 Hz, H-10a), 4.09 (d, 1 H,
H-10b), 3.95 (d, 1 H, H-30), 3.85 (dd, 1 H, J5,6a 7.3,
J6a,6b 11.9 Hz, H-6a), 3.79 (dd, 1 H, J5,6b 5.9 Hz, H-
6b), 2.17, 2.13, 2.12, 2.11, 2.04, 2.01(each s, each 3
H, OAc). Anal. Calcd for C24H32ClN3O15: C,
45.18; H, 5.06; N, 6.59. Found: C, 44.84; H, 5.35;
N, 6.37.
0
0
3-O-Acetyl-30-azido-30-deoxy-10,2:4,6-di-O-iso-
propylidene-60-O-p-toluenesulfonylsucrose (25).Ð
To a solution of 24 (330 mg, 0.674 mmol) in
pyridine (3.3 mL) were added p-toluenesulfonyl
chloride (257 mg, 1.35 mmol) and DMAP (20 mg,
0.16 mmol) at 0 ꢀC under Ar atmosphere. After the
mixture was stirred at room temperature for 12 h, it
was poured into aq NaHCO3 and extracted with
EtOAc. The organic layer was washed with brine,
dried (MgSO4), and evaporated. Flash chromato-
graphy (2:1 hexane±EtOAc) gꢀave 25 (382 mg, 88%)
30-Amino-60-chloro-30,60-dideoxysucrose (27).Ð
To a solution of 26 (34 mg, 0.053 mmol) in dry
MeOH (2.0 mL) was added NaOMe (3.0 mg,
0.056 mmol) under an Ar atmosphere. After 5 h, Lin-
dlar catalyst (20 mg) was added to the solution,
and the mixture was stirred under 1 atm of hydro-
gen for 30 min. After insoluble material was
removed o€ by ®ltration, the ®ltrate was neu-
tralized with 0.1 M HCl, and it was lyophilized.
The residue was puri®ed by gel ®ltration and lyo-
philized to give 27 (10 mg, 52%) as a white solid:
as a colorless oil: [ꢀ]2d2 +25 (c 1.6, CHCl3); H
1
NMR (CDCl3): ꢂ 7.79 (d, 2 H, JAB 8.3 Hz, Ph),
7.36 (d, 2 H, Ph), 5.93 (d, 1 H, J1,2 3.6 Hz, H-1),
5.27 (t, 1 H, J2,3 9.6, J3,4 9.6 Hz, H-3), 4.50 (dd, 1
H, J3 ,4 8.6, J4 ,5 6.3 Hz, H-40), 4.30±4.11 (m, 3 H,
0
0
0
0
H-50, 60), 4.08 (d, 1 H, J1 a,1 b 12.5 Hz, H-10a), 3.87
(dd, 1 H, J5,6a 4.6, J6a,6b 9.9 Hz, H-6a), 3.80 (d, 1
H, H-2), 3.71 (dd, 1 H, J5,6b 4.9 Hz, H-6b), 3.62
(dd, 1 H, J4,5 9.2 Hz, H-4), 3.41 (d, 1 H, H-10b),
3.27 (d, 1 H, H-30), 2.46 (s, 3 H, Me), 2.07 (s, 3 H,
[ꢀ]2d2 +74ꢀ (c 0.72, H2O); H NMR (D2O): ꢂ 5.44
1
0
0
0
0
(d, 1 H, J1,2 3.6 Hz, H-1), 4.46 (dd, 1 H, J3 ,4 9.2,
J4 ,5 9.2 Hz, H-40), 4.21 (dt, 1 H, J5 ,6 a 5.3, J5 ,6 b
5.3 Hz, H-50), 4.03 (d, 1 H, H-30), 3.95±3.74 (m, 6
H), 3.75 (t, 1 H, J2,3 9.9, J3,4 9.9 Hz, H-3), 3.62 (dd,
0
0
0
0
0
0

114
H. Kakinuma et al./Carbohydrate Research 312 (1998) 103±115
1 H, H-2), 3.48 (t, 1 H, J4,5 9.9 Hz, H-4); 13C NMR
(67 MHz, D2O): ꢂ 105.6, 92.7, 82.8, 78.0, 73.4, 73.3,
71.7, 70.3, 61.8, 61.2, 60.8, 46.0. Anal. Calcd for
C12H22ClNO9: C, 40.06; H, 6.16; N, 3.89. Found:
C, 39.73; H, 5.97; N, 3.81.
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tion from 1060-disubstituted sucrose analogs to
acceptor 2 were measured for 5±6 di€erent donor
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and a constant concentration of the acceptor 2
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We thank Dr. Y. Nakajima (Mitsui Sugar Co.
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(RIKEN) for supporting the enzyme puri®cation.

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